The present technique relates generally to image analysis designed to determine the cell cycle phase of a particular cell. More specifically, the present technique relates to automatically segmenting a cell into a cell cycle phase using three-dimensional segmentation of two-dimensional time-lapse images.
When eukaryotic cells replicate, they pass through a tightly regulated series of events known as the cell cycle. The cell cycle generally includes four phases: G1, S, G2, and M. Each phase of the cell cycle is marked by distinctive characteristics in cell morphology and total DNA content as the DNA is replicated and the cell splits into two daughter cells.
In basic research and in drug discovery work, valuable information can be obtained by understanding how an agent affects the growth and division of cells. Often, this information gives some indication of the mechanism of action associated with the compound. For example, a particular class of drugs or genetic manipulations may arrest cell growth at the G2 stage (second gap phase) and may act via a particular set of mechanisms or actions. Another class of drugs or genetic manipulations may arrest cells while in mitosis, and thus may act via a different mechanism. The ability to quickly determine whether a population of cells is blocked or arrested in G2 or mitosis (or some other stage) provides a valuable tool in assessing the mechanism of action of an uncharacterized compound that has been tested on the population of cells. This is particularly useful in the study and treatment of cancer, since it is desirable to identify compounds that block the replication of rapidly proliferating cancer cells without perturbing normal cells. Further, it is also useful to determine if an uncharacterized compound has the effect of increasing progression through the cell cycle, because such a compound may be potentially carcinogenic.
Typically, cell cycle progression is assessed by image analysis of fluorescent cellular images. Such analysis may involve staining cells with a nuclear dye, generally a fluorescent dye, to identify cell nuclei to provide a reference point for cell segmentation and other image analysis procedures. The common use of fluorescent dyes presents barriers for long-term cellular imaging. For example, nuclear dyes rely on binding in one way or another to DNA in order to adequately stain the cells. While such dyes may be suitable for fixed cell assays or for live assays of short duration (e.g. several hours), fluorescent dyes have toxic side effects that prevent long-term studies. If cells are stained with a nuclear dye and cultured for extended periods, they die, either because they cannot replicate their DNA when it is intercalated with dye, or because the dye interferes with chromosome segregation during mitosis.
An alternative method to stain nuclei and keep cells alive through more than one cell cycle is to engineer the cell to express a fluorescent protein such as Green Fluorescent Protein (GFP) that is coupled to a nuclear localization sequence so the protein acts as a nuclear stain. Although GFP staining does not interfere with DNA replication in the same manner as intercalating fluorescent dyes, this approach involves genetic manipulation of the cells. This approach also does not distinguish among the various phases of the cell cycle. More specifically, a GFP protein marker may differentially stain cells in various phases of mitosis (e.g. prophase, metaphase, anaphase, and telophase), but the marker does not distinguish among the three phases of interphase: G1, S, and G2.
In addition to staining methods that do not interfere with long-term progression through the cell cycle, there is a need for rapid and automatic assessment of such stained cells. As much basic research and drug discovery is conducted on a “high-throughput” basis, manual assessment of stained cell images for long-term studies is intractable. For example, a cell assay well may have several hundred cells that may be monitored several times per hour over several days. In addition, for each agent or potential therapeutic compound studied, several wells may be prepared to provide statistically significant results. Therefore, there exists a need for techniques allowing the reliable, accurate, and automatic determination of cell cycle progression.